Cell Reprogramming
Keys Of Reprogramming *Find and culture the minimum number of cells needed *Find and culture the optimal cell type **Usually as determined as possible *Find the best mode of implantation Chemo-attractants *In the case of skin grafts, Chemo-attracts are released naturally to mobilize BMSCs to repair damage without scarring *Nucleonin *Antithrombin III (ATB3) -> 0 - 48 Hr *HMBG1 gene expression -> 0 - 8 Hr Kinase Inhibitors *If Kinase Inhibitors spur cells to become iPSCs, then that means that the body has a natural way to make stem cells, when tissues are physically damaged, from either adult differentitated cells or able to mobilize other stem cells from local stem cell niches to repopulate the damaged tissues. Growth Factors *Affect many compnents of both growth and differentiation *Need the appropriate selection for the cell type *Too much growth factor or overexposure can lead to the poisoning of the cell culture Cell Safety Improvement *During reprogramming and Differentiation into mature tissues, DNA can be damaged **Can lead to tumor growth **Leading drawback in ineffiency *Researchers have developed a low-cost highly effective way to detect and then purge at-risk cells at an early stage in the differentiation process *Old strategies focus on separating or depleting cells after they are damaged **Expensive, difficult, and time consuming *The team pretreated cultured stem cells with a genotoxin **An agent that finds and kills cells that have gene mutations or chromosome changes *Stem cells primed with upregulation of the Puma protein *Was effective in trials and safe and didn't affect cell differentiation for iPSCs and ESCs *Shows promise to reduce risk of tumor growth after tissues are implanted into the body *This can be included in cellularized products for safety **Huge help with FDA concerns Teams Study Could Pave Way To Rejection-Free Adult Stem Cells Original Article *A German research team has generated blood-forming stem cells from iPSCs without using animal serum *Could help pave the way for rejection free adult stem cells *Animal serum can carry viruses **Can interfere with reproduction and cause many complications *They produced them in a low oxygen environment **When transplanted the cells were capable of rebuilding the blood system *This avoids the big "graft versus host" issue *Will help with clinical trials and FDA approval *Will almost definitely have to stop the use of animal serum before selling any stem cell solutions to hospitals or government Inducing Apoptosis To Increase Safety of Pluripotent Cell Derivatives Original Article *The possibility for teratomas in ESCs and iPSCs is an important problem for therapies *One option is to delete the PSCs from differentiated tissues before transplantation and leave normal cells *Etopaside treated cultures become depleted of PSCs **leaves differentiated progenitors alone *PSCs readily undergo Puma-mediated apoptosis in response to DNA damage *Doesn't cause genomic instability or loss of function *Greatly reduces the teratomagenic nature of PSCs *Would be very useful during organ recellularization or for stem cell seeded biomaterial scaffolds to reduce teratoma risk Early Reprogramming Factors Uncovered Original Article *Parp1 and Tet2 *One of the most important aspects of the reprogramming process is erasure of the somatic epigenetic pattern and the establishment of an embryonic stem cell like epigenetic pattern *These are important for the early stage of reprogramming *Parp1 and Tet2 have complementary roles in the establishment of early epigenetic marks that direct pluripotency *They looked for epigenetic modifiers that promote OSKM reprogramming of MEF *The protein Parp1 was identified as a potent inducer of reprogramming **Involved in chromatin remodeling **Increased binding at nanog and Esrrb promoter regions *Tet2 enzyme had increased interractions with the Nanog and Esrrb promoter regions during reprogramming *A loss of either Tet2 or Parp1 causes slowing or even abolition of iPSC formation *Parp1 overexpression induced Oct4 binding *OSKM directly activated them *Possibly add Parp1 and Tet2 to the growth factor mix on acellular products since OSKM can't be used but mimicked *Test if the addition of the proteins alone will induce or increase the efficiency of reprogramming Stem Cell Protectors *Even when the body mobilizes stem cells to produce billions of mature blood cells it keeps a reserve of undifferentiated stem cells *Those hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types *They are protected from over differentiation *Protected by mesenchymal cells and bone marrow monocytes *A rare population of macrophages supress HSPCs from over differentiation **Produce a substance called prostaglandins ***a-smooth muscle actin (aSMA) absorbed by HSPCs *These myeloid cells resisted radiation induced death **Help HSPCs survive chemotherapy *COX-2-derived prostaglandin E2 (PGE2) prevented HSPCs exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12 New Way To Weed Out Problem Stem Cells Making Therapy Safer Original Article *Researchers found a way to detect and eliminate potentially trouble making stem cells to help make cell therapy safer *During the differentiation process some stem cells may remain and cause tumors *The risk of tumors slows FDA approval of iPSC therapies *They pretreated the stem cells with a chemotherapeutic agent that selectively damages the DMA of the stem cells efficiently killing off the tumor forming cells **Didn't affect healty cells *Helps reduce the lab / clinic barrier *Look into journal article Stem Cell Scientists Discover Potential Way To Expand Cells For Use With Patients Original Article *Mater control gene could create a way to expand human blood stem cells (hbSCs) for clinical use *The used noncoding RNA called miRNA *They removed miR-126 **Normally it governs the expression of hundreds of other genes by keeping them silenced and keeps stem cells in a non-dividing state *The method was to introduce excess numbers of miR-126 binding sites into the stem cells by using specifically designed viral vectors **Acted like a sponge, mopping up the specific miRNA in the cells *They observed long term expansion of the blood stem cells without exhaustion or malignant transformation *Could reduce the cost of expansion *Would lower the number of stem cells needed from harvesting to treat patients Efficient, Protein-Based Method For Generating iPS Cells Original Article *Skin cells can be induced using a virus carrying 4 genes **Clinics cautious due to residual DNA and genes that have an increased risk for cancer *Only insert the proteins that those genes code for rather than the whole gene **This isn't the first time it's been done ***Has worked in the past, just not as efficiently *Standford has had unprecedented success *Viruses are more than just a Trojan Horse **Can make DNA loosen chromatin and make the DNA available for the necessary changes for pluripotency *The cell defends itself by accessing the DNA which can cause it to allow the pluripotency-inducing proteins to modify the DNA naturally *Similar to inflammation response "transflammation" *Using only proteins worked but inefficiently **Even with unrelated viruses there was a dramatic increase in effiiciency and efficacy ***Activation of toll-like receptor-3 pathway ***Faking the pathway yeilded similar results *Because it's non-integrating there is no change to the host On Nations And Inflammation: How Viruses Can Promote Cellular Reprogramming Original Article *Originally viruses were made using viral vectors *Fear of the viral vectors causing unforseen changes in the host, researchers developed cell permenant peptides (CPPs) to deliver the genes, but much less efficiently than the virus method *There is something intrinsic in using the viruses *CPPs are slower and less efficient than viral vectors **No difference if CPPs are used in conjunction with an 'empty' viral vector that carries a flourescent gene *By activity the inflammatory response through TLR3 receptors, the viruses cause epigenetic changes that put chromatin in a more open state that is conductive to iPSC formation *The response can be mimicked by using synthetic TLR3 agonists (poly I ic) **Those can increase the efficiency of CPP mediated iPSC formation **Doesn't include the risks of using viruses *Can cause activation of different receptors yield similar responses *Use an 'empty' virus or mimick the way it opens up the chromatin along with normal reprogramming factors and methods 'Fountain Of Youth' Technique rejuvenates Aging Stem Cells Original Article *Until now clinical trials using elderly patients' own cells have not been a viable option *The aged cells don't function as well as young ones *They treated a microenvironment that allows heart tissue to grow *The cell cultures are then infused with a combination of growth factors positioned in such a way within the porous scaffolding that the cells are able to be stimulated by these factors *This effectively turned back the clock in the cells returning them to a robust and healthy state Body May Be Able To 'Coach" Translpanted Stem Cells To Differentiate Appropriately Original Article *To prevent teratomas researchers must give the cells a not so subtle shove toward final development before tranplanting *Using the body to give these signals that direct differentiation could speed FDA approval **Reduces time needed to be in the lab before transplantation ***Will increase efficiency and reduce cost *They hope to use mechanical signals from the seeded scaffold to mimick the natural structures *They removed a chunk of bone and implanted a scaffold coated with BMP-2 and seeded it with 1 million stem cells **The human cells repaired the defect **The scaffold was replaced with normal bone *iPSCs healed 96% *ESCs healed 99% *Only 2 out of 42 developed teratomas from ESCs during bone growth *Both formed many teratomas in the kidney The Functions Of microRNAs In Pluripotency And Reprogramming Original Article *PSCs supress a distinctive set of miRNAs **Many work together and target the same thing *hPSCs upregulate hsa-miR-306, hsa-miR-166, hsa-miR-67, hsa-miR-195, and hsa-miR-200 families *hsa-let-7 family is expressed less than normal *microRNAs have been shown to enhance the conversion of fibroblasts to neurons and cardiomyocytes *derepression of Lin28 inhibits procession of let-7 which results in enhanced reprogramming *lentiviral expression of hsa-miR-302 and hsa-miR-367 was reported to reprogram fibroblasts 2 orders of magnitudehigher efficiency than the OSKM transcription factor based method **Only reported method that doesn't use vector based gene transfer, oncogenic genes, transcripts, or proteins *Same miRNAs are responsible for shortening the cell cycle *Several miRNAs have been shown to promote the mesenchymal to epithelial transition (MET) **hsa/mmm-miR-205, hsa/mmm-miR-200, hsa/mmm-miR-302, hsa/mmm-miR-372 *hsa-miR-17, hsa-miR-106 inhibits apoptosis in cancer cells Molecular Mechanisms Underlying Stem Cell Reprogramming Decoded Original Article *The process of generating iPSCs is very inefficient and costly *It can take a month to fully reprogram somatic cells *As few as 1 in 1000 cells that take up all four factors successfully convert *At 48hr after initiation of iPSC reprogramming the factors tended to bing gene regulatory sequences called enhancers **far removed from the genes that they regulate *Suggests that OSK serve as 'pioneer factors' that open up closed chromatin structures on the DNA **faciliates the reprogramming of process by opening up the the target genes so that they can be read by mRNA *Large chunks of the genome were physically resisting the factors from entering 'refractory sequences' *Usually marked by a histone called H3K9mo3 *When the team blocked the enzymes that create that modification they significantly accelerated reprogramming *How did they block the factors? *Integrate into Reprogramming Mix Skin Cells Reprogrammed Into Brain Cells Original Article *Scientists transformed skin cells into functional brain cells *Could lead to better models for testing Alzheimer's drugs **Afflicts 5.4 million people in the United States and expected to triple by 2050 *Transfered Sox2 in mouse and human skin cells *Within days the skin cells transformed into early stage brain stem cells called iNSCs *Within a month the neurons developed into neuronal networks *Many drugs fail in clinical trial because they don't accurately predict the drug's effect on the brain *iNSCs could help assess their efficacy and safety **Reduces risks and resources used in human trials *By avoiding the iPSC state it prevents rogue iPSCs *Saw the neurons integrate into the mouse's brain with no tumor growth *Current individual treatments can cost $200,000 *Good for making single cell types from skin by skipping PSC stage, but wouldn't work for organs or tissue that have multiple cell types Scar Tissue Turned Into Heart Muscle Without Using Stem Cells Original Article *Scientists have shown the ability to turn scar tissue that forms after a heart attack into functional heart tissue **Eliminates the need for a stem cell transplant *They use microRNAs to trigger the conversion of scar tissue into cadiac tissue *Could lead to a new way for treating many of the 23 million people world wide who suffer from heart failure **Could be adapted for brain, kidneys, and other organs *microRNAs are master regulators controlling the activity of multiple genes *They reprogrammed fibroblasts to become cardiomyocytes *Shown to work not only in the lab, but also in the body of a mouse **A major step to becomming a realistic therapy *Bypasses the need to implant stem cells **Along with all of the problems and complications **Also requires less time money, and equiment *Eliminates the genetic alterations to cells that are required when generating iPSCs